Evaluating the association of bone morphogenetic protein 4-V152A and SIX homeobox 6-H141N polymorphisms with congenital cataract and microphthalmia in Western Indian population.

Background: Congenital cataract and microphthalmia are highly heterogeneous congenital eye disorders that affect normal vision. Although mutation in several genes has been shown to cause congenital cataract and microphthalmia, genetic studies associating single-nucleotide polymorphisms with these conditions is scarce. Hence, the present study aims to investigate the association of bone morphogenetic protein 4 (BMP4)-V152A (rs17563), and SIX homeobox 6 (SIX6)-H141N (rs33912345) polymorphisms with congenital cataract and microphthalmia in Western Indian cohorts. Materials and
Methods: BMP4-V152A and SIX6-H141N were genotyped in 561 participants comprising of 242 congenital cataracts, 52 microphthalmia, and 267 controls using polymerase chain reaction (PCR) and allele specific oligonucleotide (ASO)-PCR method, respectively.
Results: The frequency of BMP4- 152A was found to be significantly different between the cases and controls (Odds ratio (OR) 95% confidence interval [CI] = 1.4 [1.03-1.76], P = 0.0275). The frequency of BMP4- 152AA genotype was found to be significantly higher in congenital cataract cases as compared to controls (OR [95% CI] = 2.1 [1.14-3.67], P = 0.0154. The V-N haplotype of BMP4-V152A and SIX6-H141N was found to have a protective effect toward congenital cataract (OR [95% CI] = 0.72 [0.56-0.94], P = 0.0163) and microphthalmia (OR [95% CI] = 0.63 [0.40-1.01, P = 0.0541). Conclusions: The BMP4- 152AA genotype might play role in the causation of congenital cataract, whereas BMP4-SIX6 V-N haplotype might play a protective role toward the development of congenital cataract and microphthalmia.

Introduction

Congenital cataract and microphthalmia are the major cause of childhood blindness with a prevalence of 1–6 and 1.7/10,000 live births, respectively.[12] Congenital cataract is characterized by lens opacity present at birth while microphthalmia represents a globe with a total axial length at least two standard deviations (SDs) smaller than the mean for age. They are highly heterogeneous and may occur either jointly or in isolation or as part of a syndrome.[3456]

Several genetic and environmental regulators are known to play roles in the causation of microphthalmia as well as cataract onset, progression, and maturation.[37] However, genetic factors have been suggested to play important role in these processes.[8] Several genetic loci and mutation in genes encoding crystallins, membrane transport and channel proteins, cytoskeletal proteins, and transcription factors (TFs) have been identified in patients with congenital cataract and microphthalmia.[34910111213141516]

TFs of the homeodomain family essentially contribute to the overall development of eye. In addition to congenital cataract and microphthalmia, mutation in TF genes have also been shown to cause wide range of eye disorders such as anophthalmia, glaucoma, and Peter's anomaly by ample of studies carried out in India as well as overseas.[171819] This led us to hypothesize that polymorphisms in these TF genes might also influence the risk toward such eye disorders. Hence, we investigated the potential association of polymorphisms V152A and H141N in TF genes bone morphogenetic protein 4 (BMP4) and SIX homeobox 6 (SIX6), respectively, with congenital cataract and microphthalmia in Western Indian population. Both these gene maps to chromosome 14 and have been shown to play a very important role during fetal eye development, such as BMP4 in lens induction, iris, and retina development.[202122] and SIX6 in lens, retina, and pituitary development.[2324] To the best of our knowledge, these polymorphisms have never been tested for association with congenital cataract and microphthalmia. This study gives an insight on the role of BMP4-V152A and SIX6-H141N polymorphisms in diagnostics and risk prediction toward the development of congenital cataract and microphthalmia.

Materials and Methods

Study subjects

This study was approved by the Institutional Review Board (IEC/ICIRC/2011-V8) and adhered to the tenets of the Declaration of Helsinki. Informed consent was obtained from all the study participants after discussing the nature of the present study. A total of 561 participants were recruited, out of which, 242 were congenital cataracts, 52 were microphthalmia, and 267 were age-matched controls. Congenital cataracts were examined using a slit-lamp biomicroscope or operating microscope and classified based on the zone and morphology of opacification. Microphthalmia was diagnosed as an eye with corneal diameter < 10 mm and mean axial length < 19 mm.[325] The inclusion criteria consisted of cases with and without a family history, having either isolated congenital cataracts and microphthalmia diagnosed between the ages of 0.1–1 years. The control participants were without any family history of ocular diseases. Cases with traumatic cataracts, TORCH infections, chromosomal abnormalities such as Down's syndrome, and other systemic disease such as heart defects and inborn errors of metabolism were excluded from this study.

Genotyping

Genomic DNA was extracted from peripheral blood using the salt-extraction method.[26] The BMP4-V152A and SIX6-H141N polymorphisms were selected for genotyping based on their minor allele frequency. Genotyping of BMP4-V152A and SIX6-H141N was carried out using polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) and allele specific oligonucleotide (ASO)-PCR, respectively. PCR-RFLP and ASO-PCR primers were designed using primer3.0 (http://primer3.ut.ee/)[27] and WASP (http://bioinfo.biotec.or.th/wasp)[28] online program, respectively. BMP4 was amplified using primers BMP4-FW: 5'-TGCTTATTTTCCCCCAGTAGGT-3’ and BMP4-RV: 3'-GGCGCCGGCAGTTCTTATTCTT-5’ that gives an amplicon of length 704 bp. The BMP4-V152A polymorphism was analyzed using RestrictionMapper version 3.0 (www.restrictionmapper.org/) and was found to create a loss of HphI restriction enzyme site for C allele. The SIX6-H141N was genotyped using a set of three primers consisting of a wild type forward primer 5'-CTTCAAGGAGCGCACGCGAA-3', a mutant forward primer 5'-CTTCAAGGAGCGCACGCGAC-3', and a common reverse primer 3'-GGCTCTTAACCCAAGACAG-5’ that gives an amplicon of length 308 bp. Further, 10% of samples were randomly selected and the genotypes were confirmed by Sanger's bi-directional sequencing using BigDye® Terminator V3.1 Cycle Sequencing kit and run on the ABI 3130xl Genetic Analyzer.

Statistical analysis

The continuous variables were tested by Student's t-test and were presented as mean ± SD. Categorical variables were presented in terms of frequencies and percentages. Allele, genotype, haplotype frequencies, and Hardy–Weinberg equilibrium (HWE) were determined using SHEsis (http://analysis.bio-x.cn/myanalysis.php).[29] To check the strength of association of polymorphisms between the cases and controls, Chi-square test was done using www.socscistatistics.com/pvalues/chidistribution.aspx. Odds ratio (OR) at 95% confidence interval (CI) was calculated to check the association of alleles and genetic models using http://www.hutchon.net/confidor.htm. The P < 0.05 was considered as statistically significant.

Results

In the present study, we have recruited 242 participants diagnosed with congenital cataract, 52 participants with microphthalmia and 267 age-matched participants that served as the control after thorough clinical examination that identified them as having no congenital cataract. The cases recruited were aged between 1 and 10 years and diagnosed with isolated microphthalmia and congenital cataract with varied phenotypes such as nuclear, lamellar, posterior subcapsular cataract (PSC), membranous, sutural, and total cataracts. There was no significant age difference between the cases and controls enrolled in this study (P > 0.05). The demographic details of participants enrolled in the present study are shown in Table 1.

Table 1

Demographic details of the study groups

Demography	Controls (n=267)	CCs (n=242)	MT (n=52)
Female, n (%)	144 (53.9)	138 (57.0)	32 (61.5)
Male, n (%)	123 (46.1)	104 (43.0)	20 (38.5)
Age (mean±SD)	5.83±2.10	6.11±2.14	5.19±2.43
P	-	0.14	0.10
Isolated cataract type (%)
Nuclear	-	42 (17.4)	-
Lamellar	-	59 (24.4)	-
PSC	-	46 (19.0)	-
Membranous	-	28 (11.6)	-
Sutural	-	15 (6.2)	-
Total	-	52 (21.4)	-
MT (%)			-
Isolated	-	-	52 (100)

PSC: Posterior subcapsular cataract, MT: Microphthalmia, SD: Standard deviation, CCs: Congenital cataracts

Genotyping by PCR-RFLP of BMP4-V152A yielded six fragments (244, 203, 121, 87, 35, and 14 bp) for T allele, and 5 fragments (447, 121, 87, 35, and 14) for C allele when digested with HphI (GGTGA [N8]↓) restriction enzyme. The agarose gel electrophoresis and the corresponding electropherograms showing all the three possible genotypes for both the polymorphisms are shown in Figure 1. The allele and genotype frequencies of both the polymorphisms were in HWE in both cases and controls. The frequency of BMP4- 152A was found to be significantly different between the cases and controls (OR [95% CI] = 1.4 [1.03–1.76], P = 0.0275) [Table 2]. The frequency of BMP4-AA was found to be significantly increased in congenital cataract cases when compared to controls (OR [95% CI] = 2.1 [1.14–3.67], P = 0.0154) [Table 2]. Using recessive model also, the frequency of BMP4-AA genotype was found to be significantly different between congenital cataracts and controls (OR [95% CI] = 2.0 [1.11–3.41], P = 0.0186) [Table 3]. There was no significant difference in the allele and genotype frequencies of SIX6-H141N polymorphism between the cases and controls [Table 4]. Since BMP4 and SIX6 lie on the same chromosome (i.e., Chr. 14), haplotype analysis was carried out to check the effect of combination of alleles toward congenital cataract risk. Haplotype analysis of BMP4-V152A and SIX6-H141N showed that V-N haplotype have a protective effect toward the development of congenital cataract (OR [95% CI] = 0.72 [0.56–0.94], P = 0.0163; D’ = 0.005, r = 0.00) and microphthalmia (OR [95% CI] = 0.63 [0.40–1.01, P = 0.0541; D’ = 0.020, r = 0.00) [Table 5]. There was no significant difference in the allele and genotype frequencies of both the polymorphisms between the different congenital cataract types and controls (data not shown). After correcting the P value (P = 0.025), BMP4- 152AA genotype was found to be significantly associated with the risk toward congenital cataract while V-N haplotype was found to confer a protective effect toward the development of congenital cataract.

Figure 1

(a and c) agarose gel electrophoresis shows polymerase chain reaction-Restriction Fragment Length Polymorphism patterns for bone morphogenetic protein 4-V152A after digesting with HphI restriction enzyme and allele specific oligonucleotide-polymerase chain reaction pattern for SIX homeobox 6-H141N, respectively. (b and d) shows electropherogram confirming all the three possible genotypes of bone morphogenetic protein 4-V152A and SIX homeobox 6-H141N

Table 2

Distribution of BMP4-V152A in the study groups

Study groups	BMP4-V152A
	A (C-allele)	V (T-allele)	AA	AV	VV
Controls (n=267)	149 (0.279)	385 (0.721)	22 (0.082)	105 (0.393)	140 (0.524)
CC (n=242)	166 (0.343)	318 (0.657)	36 (0.149)	94 (0.388)	112 (0.463)
MT (n=52)	35 (0.337)	69 (0.663)	7 (0.135)	21 (0.404)	24 (0.462)
Control versus CC, OR (95% CI)	1.4 (1.03-1.76)	Reference	2.1 (1.14-3.67)	1.1 (0.77-1.63)	Reference
P	0.0275	-	0.0154	0.5500	-
Control versus MT, OR (95% CI)	1.3 (0.84-2.05)	Reference	1.9 (0.71-4.82)	1.2 (0.62-2.21)	Reference
P	0.2362	-	0.1988	0.6356	-

P<0.05 was considered as statistically significant. A: Alanine, V: Valine, CC: Congenital cataract, MT: Microphthalmia, OR: Odds ratio, CI: Confidence interval

Table 3

Dominant and recessive models for BMP4-V152A and SIX6-H141N

Study groups	BMP4-V152A		SIX6-H141N
	Dominant VV versus (VA + AA)	Recessive AA versus (VV + VA)	Dominant NN versus (NH + HH)	Recessive HH versus (NN + NH)
Controls (n=267)	140/127 (0.476)	22/245 (0.918)	69/198 (0.742)	68/199 (0.745)
CC (n=242)	112/130 (0.537)	36/206 (0.851)	78/164 (0.678)	58/184 (0.760)
MT (n=52)	24/28 (0.538)	7/45 (0.865)	17/35 (0.673)	26/26 (0.500)
Control versus CC, OR (95% CI)	0.8 (0.55-1.11)	2.0 (1.11-3.41)	1.4 (0.93-2.00)	0.9 (0.62-1.38)
P	0.1655	0.0186	0.1122	0.6951
Control versus MT, OR (95% CI)	0.8 (0.43-1.41)	1.7 (0.70-4.30)	1.4 (0.73-2.65)	0.61 (0.28-132)
P	0.4071	0.2308	0.3085	0.2084

P<0.05 was considered as statistically significant. V: Valine, A: Alanine, N: Asparagine, H: Histidine, CC: Congenital cataract, MT: Microphthalmia, OR: Odds ratio, CI: Confidence interval

Table 4

Distribution of SIX6-H141N in the study groups

Study groups	SIX6-H141N
	N (A-allele)	H (C-allele)	NN	NH	HH
Controls (n=267)	266 (0.498)	268 (0.502)	68 (0.255)	130 (0.487)	69 (0.258)
CC (n=242)	222 (0.459)	262 (0.541)	58 (0.240)	106 (0.438)	78 (0.322)
MT (n=52)	44 (0.423)	60 (0.577)	9 (0.173)	26 (0.500)	17 (0.327)
Control versus CC, OR (95% CI)	0.9 (0.67-1.10)	Reference	0.8 (0.47-1.21)	0.7 (0.47-1.09)	Reference
P	0.2083	-	0.2469	0.1207	-
Control versus MT, OR (95% CI)	0.7 (0.48-1.12)	Reference	0.5 (0.22-1.29)	0.8 (0.41-1.60)	Reference
P	0.1612	-	0.1596	0.5459	-

P<0.05 was considered as statistically significant. N: Asparagine, H: Histidine, CC: Congenital cataract, MT: Microphthalmia, OR: Odds ratio, CI: Confidence interval

Table 5

Haplotype distribution of BMP4-V152A and SIX6-H141N

Study groups	Loci
	AN	AH	VN	VH
Controls (n=534)	71.79 (0.134)	77.21 (0.145)	194.21 (0.364)	190.79 (0.357)
CC (n=484)	80.29 (0.166)	85.71 (0.177)	141.71 (0.293)	176.29 (0.364)
MT (n=104)	16.40 (0.158)	18.60 (0.179)	27.60 (0.265)	41.40 (0.398)
Control versus CC, OR (95% CI)	1.28 (0.91-1.80)	1.27 (0.91-1.78)	0.72 (0.56-0.94)	1.03 (0.81-1.33)
P	0.1598	0.1579	0.0163	0.8176
Control versus MT, OR (95% CI)	1.21 (0.67-2.16)	1.29 (0.74-2.25)	0.63 (0.40-1.01)	1.19 (0.77-1.83)
P	0.5293	0.3712	0.0541	0.4287

P<0.05 was considered as statistically significant. A: Alanine, N: Asparagine, H: Histidine, V: Valine, CC: Congenital cataract, MT: Microphthalmia, OR: Odds ratio, CI: Confidence interval

Discussion

TFs play an essential role in the regulation of gene expression and are vital for proper development and functioning of the body organs and other cellular processes. Hence, mutations in TF genes have the potential to cause a broad range of developmental disease including cancer. In the present study, we tested the association of polymorphisms V152A and H141N in BMP4, and SIX6 TF genes, respectively, with the risk of congenital cataract and microphthalmia. Although these polymorphisms have never been studied before in relation to congenital cataract and microphthalmia, they have been studied/associated with other disease phenotypes such as BMP4-V152A with tooth agenesis,[3031] cleft lip,[3233] and cutaneous melanoma,[34] and SIX6- H141N with primary-open angle glaucoma.[353637]

In our study, we found a significant increase in the frequency of BMP4-AA genotype in congenital cataracts which gives an indication that it might play a crucial role in gene function. In a case–control study, BMP4-V152A was reported to affect the BMP4 mRNA expression with an increased expression in BMP4- 152V carriers as compared to BMP4- 152A carriers which was associated with the risk of cutaneous melanoma.[34] In the developing eye, Bmp4 expression is first observed in the distal part of optic vesicle and underlying surface ectoderm and later stages become restricted to the developing lens.[38] It plays a crucial role in inducing high levels of Sox2 expression in the ectoderm and further in the induction of lens.[20] Hence, it is plausible that BMP4 mRNA expressed in individuals with BMP4-AA genotype might not be sufficient for the proper induction of lens and might lead to congenital cataract. In addition to this, our study also observed a significant increase in the frequency of V-N haplotype of BMP4-V152A and SIX6- H141N in controls as compared to congenital cataracts and microphthalmia which signifies that it might play a protective role toward the development of congenital cataract and microphthalmia.

To summarize, congenital cataract and microphthalmia is a highly heterogeneous disease, owing to diverse mutations in same or different genes. Further, our study suggests that in addition to mutation in candidate genes, there are several other modifying factors such as environmental factors, and an individual's genotype that could pose a serious risk toward the development of congenital cataract and microphthalmia.

Conclusion

BMP4- 152AA genotype might play an additive role in the causation of congenital cataract and BMP4-SIX6 ‘VN’ haplotype may confer a protective effect towards the development of congenital cataract. However, this study has a limitation of relatively small sample size (particularly microphthalmia patients), and hence to confirm the present findings, similar studies using large cohorts are warranted in different populations.

Financial support and sponsorship

This study was partially supported by grants from the Indian Council of Medical Research, Government of India (file no. 5/4/6/10/Oph./11-NCD II & file no. 5/4/6/2012-RMC).

Conflicts of interest

There are no conflicts